This invention relates to the purification of ubiquitin hydrolases having enzymatic activity in cleaving ubiquitin-protein conjugates. This invention also relates to a process for preparing ubiquitin hydrolases using recombinant methods and a process for using same to isolate polypeptides from fusions thereof with ubiquitin.
The polypeptide known as ubiquitin is highly conserved, has a molecular weight of 8,565, and contains 76 amino acid residues. It is encoded by genes that contain varying numbers of the protein sequence repeated without any stop codons between them or with other proteins. Ubiquitin is reviewed in Rechsteiner, Ann. Rev. Cell. Biol., 3: 1-30 (1987) and in Rechsteiner, M., ed., Ubiquitin (New York: Plenum Press, 1988).
Ubiquitin was first purified during studies of peptides of the thymus. Radioimmunoassays for the peptide revealed that it was found widely in plant, animal, and yeast. Goldstein et al., Proc. Natl. Acad. Sci. U.S.A. 72: 11-15 (1975). The sequence of amino acids 1-74 of thymus ubiquitin was determined by Schlesinger et al., Biochemistry, 14: 2214-2218 (1975), revealing an NH.sub.2 -terminal methionine and an arginine at position 74. The sequence was confirmed by Low et al., J. Biol. Chem., 254: 987-995 (1979). This form was later shown to be a degraded form of ubiquitin and is not active in its biological function. The active form is the 76 amino acid form. Wilkinsion and Audhya, J. Biol. Chem., 256: 9235-9241 (1981).
It has been found that ubiquitin is involved in the energy-dependent degradation of intracellular proteins. Ganoth et al., J. Biol. Chem., 263: 12412-12419 (1988). Evidence exists that in eukaryotes, covalent conjugation of ubiquitin to the proteins is essential for their selective degradation. Finley and Varshavsky, Trends Biochem. Sci., 10: 343-346 (1985) and Finley et al., Cell, 37: 43 (1984).
Isopeptidases have been identified that are unique for eukaryotes. They are found to cleave in vitro an amide bond formed between a ubiquitin Gly-COOH terminal and epsilon-NH.sub.2 group of lysine on other polypeptides. For example, an isopeptidase was identified that cleaves the linkage between ubiquitin and lysozyme to yield free lysozyme. Hershko et al., Proc. Natl. Acad. Sci. U.S.A., 81: 1619-1623 (1984). An isopeptidase was also detacted in reticulocyte extracts that cleaves ubiquitin-histone 2A conjugates, with the release of undegraded histone. Andersen et al., Biochemistry, 20: 1100-1104 (1981), Kanda et al., Biochim, Biophys. Acta, 870: 64-75 (1986), Matsui et al., J. Cell Biol., 95: (2) PA82 (1982), and Matsui et al., Proc. Natl. Acad. Sci. U.S.A., 79: 1535-1539 (1982). See also Hershko et al., Proc. Natl. Acad. Sci. U.S.A., 77: 1783-1786 (1980) and Haas and Rose, Proc. Natl. Acad. Sci. U.S.A., 78: 6845-6848 (1981).
Isopeptidase was purified 175-fold from calf thymus by ionexchange chromatography, gel filtration, and affinity chromatography on whole histone and histone H2A Sepharose. Kanda et al., J. Cell Biol., 99: (4), PA135 (1984). This purified isopeptidase was found to be specific to the epsilon-(glycyl)lysine linkage in structural chromatin protein A24.
When the isopeptidase was purified, it was found to exist in growing Chinese hamster cells as two major forms having molecular weight 250,000 and 34,000, but was found to be present in human erythrocytes and calf thymus only in the 250,000 molecular weight form. These two forms of enzyme were found to be distinct from one another, in that the degradation of the large form did not result in the appearance of the smaller form. Matsui, J. Cell Biol., 105: (4 Part 2), 187A (1987). The author suggests that the large form is a stable constitutive enzyme and that the small form with a rapid turnover rate is linked to the metabolic pathway of growth-related ubiquitin-protein conjugates. Isopeptidase activity of 30 kDa on silver-stained SDS-PAGE (called carboxyl-terminal hydrolase) was also identified in human red blood cells. Pickart and Rose, J. Biol. Chem., 260: 7903-7910 (1985). The same enzyme may be involved in the cleavage as is involved in processing the ubiquitin-protein fusions. Pickart and Rose, J. Biol. Chem., 261: 10210-10217 (1986). This enzyme was formerly called ubiquitin carboxyl-terminal esterase because it was found to hydrolyze ubiquitin esters of small thiols. Rose and Warms, Biochemistry, 22: 4234-4237 (1983).
An activity of processing protease is reported in WO 88/02406 published Apr. 7, 1988, in the context of designing or modifying protein structure at the protein or genetic level to produce specified amino termini based on introducing the use of artificial ubiquitin-protein fusions.
Ubiquitin aldehyde was found to form strong complexes with most hydrolases, e.g., the major ubiquitin-protein hydrolase of greater than 200 KDa, a small 30 KDa cationic hydrolase, and the major hydrolase of 30 KDa that acts on small molecule conjugates of ubiquitin. Rose et al., Fed. Proc., 46: (6), 2087 (1987). It was concluded that ubiquitin hydrolases, in addition to being important in rescuing ubiquitin from traps with small nucleophiles, are necessary for recycling ubiquitin from protein conjugates that are only slowly degraded.
Recent analysis of the genes encoding ubiquitin from various organisms by molecular genetic techniques has shown that ubiquitin is synthesized as a polyubiquitin precursor with multiple, contiguous stretches of ubiquitin sequences or as a protein fusion in which ubiquitin is located at the N-terminal domain of a larger protein. Ozkaynak et al., The EMBO J., 6: 1429-1439 (1987); Lund et al., J. Biol. Chem., 260: 7609-7613 (1985). The last copy of the ubiquitin sequence in the polyubiquitin gene is usually followed by one amino acid extension after the unique Arg-Gly-Gly terminus. Ozkaynak et al., supra.
Another discovery regarding cleavage of ubiquitin-protein conjugates revealed that when a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein was expressed in yeast, ubiquitin was cleaved from the fusion protein, yielding a deubiquitinated beta-galactosidase. This endoproteolytic cleavage was found to take place regardless of the nature of the amino acid residue of beta-gel at the ubiquitin-beta-gal junction, with one exception. Bachmair et al., Science, 234: 179-186 (1986). It was also found that different residues could be exposed at the amino-termini of the otherwise identical beta-gal proteins. These authors suggested that the same protease, as of then uncharacterized biochemically, was responsible both for the conversion of polyubiquitin into mature ubiquitin and for the deubiquitination of the nascent ubiquitin-beta-gal protein.
Different investigators detected a proteolytic activity that converted the polyubiquitin to ubiquitin when a coupled in vitro transcription/translation system was employed. Agell et al., J. Cell Biol., 105: (4 pt 2), 82a (1987) and Agell et al., Proc. Natl. Acad. Sci., U.S.A., 85: 3693-3697 (1988). The polyubiquitin processing activity was partially inhibited by ubiquitin aldehyde, a known inhibitor of ubiquitin hydrolase. A purified preparation of this proteolytic activity was found to be inactive, with further purification of the putative protease then reported to be in progress.
At the American Chemical Society meeting on Sep. 26, 1988, chiron Corp. disclosed that fusion of a gene that has proven difficult to express directly to a synthetic gene for yeast ubiquitin has allowed high-level intracellular production of the desired protein as a mature polypeptide, cleaved in vivo by an endogenous yeast protease. See 1988 ACS Abstract Book, Abs. No. 34, P. J. Barr et al., "Production of Recombinant DNA Derived Pharmaceuticals in the Yeast Saccharomyces cerevisiae."
Regarding the purification of substances using an irreversible step such as cleavage, it was reported that fragments of proteins can be separated by charge or size in one dimension and then a reagent used to alter the protein fragments irreversibly for visualization in a second dimension. The objective of this work was to obtain amino acid sequence from the protein. Hartley et al., Biochem. J., 80: 36 (1961).
In addition, it is known to recover and purify a protein from its fusion product with an "identification" peptide. EP 150,126 published Jul. 31, 1985, equivalent to U.S. Pat. No. 4,703,004. In this process a hybrid polypeptide is synthesized with the identification peptide fused to a desired functional protein at the C-terminus of the identification peptide. The linking portion of the identification peptide is cleaved at a specific amino acid residue adjacent to the functional protein by using a sequence-specific proteolytic enzyme or chemical agent. The hybrid polypeptide is purified by affinity chromatography using an immobilized ligand specific to the antigenic portion of the identification peptide. The protein is then cleaved from the isolated hybrid polypeptide with an appropriate proteolytic agent to release the mature functional protein.
Recovery of a product from its fusion using an identification peptide linker or antibody is also disclosed. EP 35,384, published Sep. 9, 1981, and U.S. Pat. No. 4,732,852, issued Mar. 22, 1988. Moreover, recombinant production of polypeptides as fusion products with a charged amino acid polymer, separating the fusion product from contaminants based on the properties of the polymer, and cleaving the polymer from the fusion product using an exopeptidase has been reported. U.S. Pat. No. 4,532,207, issued Jul. 30, 1985.
The major problem associated with cleaving fusion proteins produced by recombinant means has been the lack of specific cleaving agents to remove the fusion protein moiety from the product protein in an exact and consistent manner. Chemical agents such as cyanogen bromide or hydroxylamine, or specific proteases such as Factor Xa or collagenase, that are used generally to achieve cleavage typically are only commercially practical in a limited number of protein fusion cleavages.
For example, if the specific amino acid that is required for the cleavage of a fusion protein (such as methionine for cyanogen bromide) is present internally in the amino acid sequence of the desired protein product, the product will be clipped internally as well as cleaved from the fused polypeptide. For this reason and other reasons, the cleaving agents are generally specific only for one protein product. In addition, the cleavage itself may leave extra amino acid residues on the product protein. Furthermore, almost all of the cleaving agents require extra recovery steps to purify the more complex mixture that is generated after cleavage.
Accordingly, it is an object of the present invention to provide a ubiquitin hydrolase that is purified to a sufficient degree that it can be sequenced.
It is another object to provide quantities of a ubiquitin hydrolase useful for commercial purposes by using recombinant means to produce the hydrolase, free of source proteins.
It is still another object to provide a procedure for obtaining a heretofore unidentified yeast ubiquitin hydrolase.
It is another object to provide a method for producing and purifying mature polypeptides, the method being characterized by removing the fusion protein moiety from the product moiety specifically and efficiently, by reducing the number and complexity of fusion recovery steps, and by obtaining precise and reproducible cleavage of the product free of extra unwanted terminal amino acid residues.
These and other objects will be obvious to those of ordinary skill in the art.